1. Field of the Invention
The present invention relates to lung cells having a novel phenotype, morphology, and immunoprotectant properties. The invention further relates to cell lines derived from such cells and methods for producing such cells, both in vivo and in vitro. The invention additionally relates to expression products of such cells and cell lines.
2. Background of the Technology
Genetic information in living creatures is contained in deoxyribonucleic acid (DNA) which is the chemical that makes up genes. Formed from long chains of xe2x80x9cnucleotide basesxe2x80x9d, genes are the building blocks of life. The nucleotide bases, guanine (G), cytosine (C), adenine (A), and thymidine (T), bond together into long chains that provide the recipe for what our bodies make. Genes dictate the production of chemicals (proteins) that make our bodies function and create our appearance, such as our sex and our hair, eye, and skin colors. Genes, however, can also be missing, defective, or not fully operational which can lead to serious medical problems. Certain medical problems that are genetic in origin are cystic fibrosis (a severe disease that commonly manifests itself in chronic lung problems), adenosine deaminase deficiency (xe2x80x9cADA deficiencyxe2x80x9d, which results in a nearly nonfunctioning immune system in a patient), xcex11-antitrypsin deficiency (xe2x80x9cAAT deficiencyxe2x80x9d, which results in liver dysfunction and emphysema style malfunction in the lungs), among others.
The recipe, mentioned above, stems from the fact that genes xe2x80x9ccodexe2x80x9d for, and xe2x80x9cexpressxe2x80x9d, proteins within cells. That is, the cellular machinery within a cell reads the genetic code that is present within the cell and manufactures (expresses) the appropriate protein. The resulting proteins are used in a variety of different processes in the body. Such proteins can be used in xe2x80x9csignalingxe2x80x9d the cells to do something, used by cells to accomplish a task, or the like. Where a gene is missing or defective, the cells are incapable of properly functioning in signaling, accomplishing their task, or the like. Where a gene is underexpressed, i.e., makes too little protein, there may be insufficient quantities of the protein present in the body to provide proper function or the like. In other cases a gene may be xe2x80x9coverexpressedxe2x80x9d, i.e., too much protein is made, which may lead to undesirable consequences. It is this latter case that is believed to play a role in certain cancers, for example, oncogenes are genes that become overexpressed and appear to play a role in the development of certain cancers.
Gene therapy is a therapeutic approach in which a disease or genetic defect in a patient is treated or corrected through providing a xe2x80x9cforeignxe2x80x9d gene that is either missing, defective, underexpressed, or will help treat the disease. The potentials of gene therapy are, therefore, enormous. In theory, many diseases can be treated or cured through the use of gene therapy.
There are many gene therapeutic approaches that are currently being studied, either preclinically, in animals, or clinically, in human patients. One of the earliest gene therapeutic approaches that was tried (in mid-1991) was the correction of ADA deficiency. ADA deficiency is an enzymatic deficiency. In ADA deficiency, patients are susceptible to virtually any infection or diseasexe2x80x94a cold, for example, can potentially be fatal. Suffers from ADA deficiency are forced to live their usually very short lives in an isolation chamber (i.e., a bubble baby).
In the gene therapeutic approach for treating ADA deficiency, in a process known as xe2x80x9cex-vivoxe2x80x9d gene therapy, particular immune cells from patients were taken from their bodies and a foreign ADA gene was inserted into the genetic material of the cells using a xe2x80x9cretrovirusxe2x80x9d (a virus that inserts itself into the DNA of a cell). Such xe2x80x9ctransducedxe2x80x9d or xe2x80x9ctransgenicxe2x80x9d cells were then capable of producing the protein adenosine deaminase. The transduced cells were reintroduced into the patients. It was recently reported that one of the patients, so treated, appears to be producing about 25% of the normal levels of ADA, while another of the patients appears to be producing only about 1% of the normal levels. Generally, in enzymatic type deficiencies, such as ADA deficiency, if one can restore approximately 10% of normal production of protein, the patient should experience generally normal function. Thus, it appears that success was achieved in one of the two patients. See New York Times; p. 422, (Oct. 20, 1995), the disclosure of which is hereby incorporated by reference.
Cystic fibrosis (CF) is a genetic disease that afflicts infants in approximately 1 of every 2000 or 9000 births, depending on race. In CF, patients experience severe xe2x80x9cendocrinexe2x80x9d misfunction. The endocrine system includes all of the cells and organs involved in the secretion of substances, particularly of hormones, in the body. One of the significant symptoms of CF, and a leading cause of death therefrom, is chronic lung problems, including, a thick mucous that fills the lungs, infections, and persistent coughing. In addition, the lungs of the patient with CF are susceptible to repeated bacterial infections which ultimately lead to lung damage and respiratory failure.
A gene, the CFTR gene, has been used in human clinical trials in an effort to restore lung function in patients suffering from CF. Some success has been achieved, however, as in many therapeutic procedures, where a foreign protein or gene is administered to a patient, the body often recognizes such materials as foreign and mounts an attack on the foreign materials, called an immune response.
An immune response is visible in situations where a cut becomes infected with bacteria, the cut swells, becomes red and inflamed, becomes warm, may have pus or the like. In this process, the body has recognized the presence of foreign bacteria and has mounted an attack on it. Further, the body has a system for remembering the foreign bacteria (xe2x80x9cmemory cellsxe2x80x9d) so that, in the future, if exposed to the bacteria again, it can mount its attack more quickly.
The immune system, and the response that the immune system mounts, is very useful in protecting our bodies against infection and many diseases. However, the very system that assists us in staying healthy, is often an obstacle to the delivery of genes to a patient. When genes are attacked by the body""s immune system, the function of the gene (the gene""s ability to produce the desired protein) is often destroyed.
It has been proposed that prenatal fetuses (in utero, prior to birth) may not possess as significant an immune response to foreign genes. It is thought that mammals, at such a stage of development are xe2x80x9cimmunotolerant.xe2x80x9d Immunotolerant essentially means that the immune systems of such animals will not so readily attack foreign genes. This belief is founded on the principal that prenatal fetuses are exposed to multiple foreign genes from the xe2x80x9cmotherxe2x80x9d in addition to the fact that the immune system in the infant is still developing.
In addition to their potential immunotolerance, prenatal fetuses offer yet another advantage over more mature mammals. This advantage is that, in theory, certain genetic defects that compromise the health of the infant upon birth can be treated prior to delivery. CF is an example; chronic lung infections can result in irreversible lung damage. Through providing the CF gene to an infant prior to their delivery from the uterus, the lung problems could potentially be avoided.
Thus, was suggested that prenatal infant mammals would be a good target for gene therapy. Indeed, based on this postulate, several groups have tried to deliver foreign genes to prenatal animals. Such groups injected foreign genes into the amniotic fluid surrounding a prenatal infant mammal. It was expected that the gene would be breathed in and swallowed by the infant as it begins xe2x80x9cpracticingxe2x80x9d using such muscles. The groups initially observed positive results; the injected gene was expressed in certain tissues in the infant mammal. In contrast to the predictions related to the immunotolerance of the prenatal infants, however, each of the groups who have tried to deliver foreign genes to such infants, in utero, have observed a significant immune response to the foreign genes that were delivered. The immune response was such that the activity of the foreign gene was destroyed within a relatively short time after delivery. See McCray et al. xe2x80x9cAdenoviral-mediated gene transfer to fetal pulmonary epitheliain vitro and in vivoxe2x80x9d J. Clin. Invest. 95:2620-2632 (1995); Kolberg xe2x80x9cRAC tiptoes into new territory: in utero gene therapyxe2x80x9d J. NIH Res. 7:37-39 (1995), the disclosures of which are hereby incorporated by reference.
In co-pending U.S. patent application Ser. Nos. 08/550,918 and 60/008,161 each filed on Oct. 30, 1995, we described the successful rat in utero delivery and long-term expression of two genes: the lac Z reporter gene (Lac Z) and the cystic fibrosis transmembrane conductance regulator gene (CFTR). We delivered the gene at a time when the lungs were developing and the budding bronchioles were lined with pleuripotential stem cells. Each of the genes were taken up by the cells and expressed for extended periods of time (i.e., for as long as we monitored the animals). No evidence of immune response was noted. In the case of the CFTR gene, moreover, we observed a phenotypic and morphological alteration of the cells. The altered cells appeared to secrete or excrete certain glycoconjugates and lipids.
In earlier work, Whitsett et al. (Nature Genetics 2:13-20 (1992)) prepared transgenic mice designed to over-express CFTR protein products. Mice were transduced with hCFTR gene under the control of a 5xe2x80x2 transcriptional element of the human surfactant protein C gene, which contains lung specific promoter-enhancer elements. While Whitsett and colleagues observed expression of the CFTR gene in the mice, review of the photomicrographs and figures in the paper do not indicate phenotypic or morphological alterations to the lung cells of the animals. Secretion or excretion of mucous like glycoconjugates in response to in vivo incubation of lung tissues with dirhamnolipid (a toxin from Pseudomonas aeruginosa) has been described by Fung et al. Am. J. Resp. Cell Mol. Biol. 12:296-305 (1995).
In view of the phenotypic and morphological alterations and apparent excretory or secretory function that we observed in CFTR expressing rodent lungs, in contrast to the work described above, we expected that the cells might possess new function and lead to a better understanding of CF disease and lung immunology in general. For, in addition to the desirability of providing a method to conduct gene transfer in a prenatal infant to correct genetic defects in the infant prior to birth, it would be desirable to understand the function of genes in early fetal cellular development and differentiation. Moreover, owing to some of the potential constraints imposed in utero gene delivery and transfer, it would be desirable to develop therapeutics based upon such elucidation which can be administered to, and operative within, infant and adult patients alike.
Cystic Fibrosis
Cystic fibrosis is manifested in a variety of ways, depending largely upon the particular variant gene present in an individual. The disease is described extensively in Cystic Fibrosis: Current Topics by J. A. Dodge (Contributor), D. J. H. Brock (Editor), J. H. Widdicombe (Editor) John Wiley and Sons 1996. Recent reviews include Rosenstein and Zeitlin (1998). Manifestations relate to the disruption of exocrine function of the pancreas but also to intestinal glands (meconium ileus), biliary tree (biliary cirrhosis), bronchial glands (chronic bronchopulnonary infection with emphysema), and sweat glands (high sweat electrolyte with depletion in a hot environment).
(Stern 1997) has roughly correlated the amount of functional CFTR produced and the phenotype. Pancreatic exocrine deficiency is at one extreme, with less that 1% of normal CFTR function, followed by progressive pulmonary infection ( less than 4.5%); demonstrable sweat abnormality ( less than 5%); congenital absence of vas deferens; no known abnormality ( greater than 10%).
The severity of pancreatic deficiency is also correlated with susceptibility to infection (Tummler, Bosshammer et al. 1997).
Target Organs
The severity of disease varies in different organs. CF disease severity in different organs and species may relate to the expression of an xe2x80x9calternativexe2x80x9d plasma membrane Cl- conductance. For example, an alternative conductance has been detected in epithelia of organs from CF mice that exhibit a mild disease phenotype (airway, pancreas) but not in epithelia with a severe phenotype (small, large intestine). (Clarke, Grubb et al. 1994)
Many individuals with congenital bilateral absence of the vas deferens (CBAVD) have CF mutations on one or both CFTR genes. They usually have no respiratory or pancreatic abnormalities, and the sweat chloride is highly variable (Teng, Jorissen et al. 1997; Cuppens, Lin et al. 1998).
CFTR During Development
Gaillard and others studied the distribution of CFTR in normal fetuses ranging from 7 to 39 wk of gestation. CFTR gene expression begins very early in lung development and, with differentiation of the airways, becomes confined to differentiated bronchiolar epithelium. (McGrath, Basu et al. 1993); (Tizzano, O""Brodovich et al. 1994); (Gaillard, Ruocco et al. 1994); (McCray, Reenstra et al. 1992; McCray, Wohlford-Lenane et al. 1992)). By seven weeks, the protein is already present in the yolk sack and in the respiratory epithelium. By the first and second trimester, CFTR mRNA is expressed throughout the human lung epithelium (McCray, Reenstra et al. 1992; McCray, Wohlford-Lenane et al. 1992). Hence, these epithelia have the capability to perform important transport functions before they are fully differentiated.
CFTR is distributed in the developing airways along the cephalocaudal pattern of maturation and differentiation of epithelial cells (Gaillard, Ruocco et al. 1994; McGrath et al, 1993; Gaillard, Ruocco et al. 1994).
Lung Development
An interspecies comparison of the development of lung structure is provided in xe2x80x9cDevelopment of Lung Structurexe2x80x9d by I. Y. R. Adamson in The Lung: Scientific Foundations, ed. by R. G. Crystal, J. B. West et al., Raven Press, Ltd. 1991
A classification of stages in lung development is based on Boyden, E. A. xe2x80x9cDevelopment of the Human Lungxe2x80x9d in Brennermatn""s Practice of Pediatrics, Vol. 4. Hagerstown, Md.: Harper and Row, 1972. Chap. 64.) Pseudoglandular period (5 to 17 weeks.
Cut sections of lung apear as acinous glands, with elaborate branching of the airway and pulmonary vasculature.
Canalicular period (13 to 25 weeks).
The bronchi and bronchiolar lumnina enlarge. Each terminal bronchiole enlongates and divides into two respiratory bronchioles.
Terminal sac period (24 weeks to birth).
New respiratory bronchioles continue to appear and two types of pneumocytes, the epithelial lining cell, can be recognized. Surfactant is begins to be produced. Extrauterine survival becomes possible as the pulmonary and vasculature develops and the epithelial lining thins.
Alveolar period (late fetal to 8 years)
Alveoli increase 6 to 8 fold to reach adult numbers by 8 years.
The Gene
CFTR expression is regulated by multiple transcriptional and translational mechanisms. Several alternatively spliced isoforms have been identified of which at least some are functional and organ specific.
Therapy
Selected compounds that activate CPTR channels are listed in Table 2
The Protein
CFTR is a member of a ubiquitous superfamily of related ATP-binding proteins, known as ABC transporters or xe2x80x9ctraffic ATPasesxe2x80x9d (Bianchet, Ko, et al. 1997). Most members are involved in active transport of small hydrophilic molecules across the cytoplasmic membrane. All these proteins share a conserved domain of some two hundred amino acid residues, which includes an ATP-binding site. They are found in both prokaryotes and eukaryotes. Members of the ABC transporter superfamily are mutated to cause diseases that include hyperinsulinemia, adrenoleukodystrophy, Stargardt disease and multidrug resistance, as well as cystic fibrosis.
The CITR channel consists of two motifs, each containing a membrane-spanning domain (MSD) and a nucleotide-binding domain (NBD), linked by a regulator domain. One MSD-NBD motif is sufficient to form a Cl- channel Ostedgaard, Rich et al. 1997). A model graphically describing a likely relationship for all of these domains is presented by Ackerman and Clapham (1997). In this model, 12 transmembrane segments span the membrane forming a pore, while the NBD1 and NBD2 domains are tethered to the pore on the intracellular side. The R, or gating domain, is tethered to the pore and to NBD1, forming a xe2x80x9cball and chainxe2x80x9d structure that functions as a phorphorylation-regulated stop valve (Ma, Tasch et al. 1996). For the Cl- channels to open, they must be phosphorylated and then exposed to a hydrolyzable nucleoside triphosphate, such as ATP (Lohmann, Vaandrager et al. 1997). Cellular phosphatases rapidly dephosphorylate the channels, inactivating them (Gadsby D C, Nairn A C. Trends Biochem Sci Nov. 19, 1994;( 11):513-518).
Protein kinase A (PKA) is necessary for phosphorylation of CFTR (McDonald, Matthews et al. 1995); neverthless, PKA alone is not sufficient to open the CFTR chloride channel in the presence of MgATP. Additional phosphorylation by protein kinase C (PKC) is required for acute activation of CFTR by PKA (Jia, et al. 1997).
Physiological Activity
CFTR is well known as a non-rectfying chloride channel, regulated by ATP and phosphate, and this function often eclipses perception of the protein""s collateral abilities (Jiang and Engelhardt, 1998). Nevertheless, CFTR comprises a complex structural and regulatory system. In these collateral roles, CFTR protein may also transport ATP (Sugita, Yue et al. 1998; Cantiello, Jackson et al, 1998); regulate outwardly rectifying chloride channels (ORCC) and epithelial sodium chloride channels (ENaC) (Ismailov, Awayda et al. 1996; Stutts, Rossier et al. 1997; Briel et al 1998); mediate vesicular trafficking (Bradbury, Cohn et al. 1994); mediate secretion of the endogenous cAMP-linked hormones VIP and secretin (Peters, van Doorninck et al 1997); associate with potassium channels (Ho, 1998); interact with the actin cytoskeleton (Cantiello, 1996); bind a Pseudomonas aeruginosa lipopolysaccharide-core oligosaccharide ligand for epithelial cell ingestion (Pier, Grout et al. 1996) and affect glycosylation. The constitutive pathway for secretion of glycoconjugates and proteins from microvesicles is intact in CF, but regulated secretion from secretory granules is defective. CF exocrine epithelia do not respond to cholinergic or adrenergic stimulation (Mills, Dorin et al. 1995; Pereira, Dormer et al. 1995).
CFTR also shows permeability to large organic anions, but only from the intracellular side of the membrane. ATP hydrolysis is required to maintain asymmetric permeability. Loss of this assymetric permeability to large organic anions may contribute to the pleiotropic symptoms seen in cystic fibrosis (Linsdell and Hanrahan 1998).
Ion Channels
The level of hydration at the surface of many tissues, particularly the lung, is partially determined by CFTR, and partially by complimentary ion channels, notably the epithelial sodium channel (ENaC) of the apical membrane. To appreciate the complex interactions that occur, it is necessary to consider the aggregate effect of all types of ion channels. About 30% of cellular energy expenditure is stored in a cellular cross-membrane sodium/potassium ion gradient. Ion channels are transmembrane protein tunnels that act as switches to relieve the ion gradient and thus release the stored energy. They are more efficient than enzymes, and they allow the flow of up to 10 million ions/second/channel. A few thousand of each type of channel/per cell is general sufficient. They are usually classified as sodium, potassium, calcium, or chloride channels. Conductance is a measure of ion flow and it is expressed as the charge/second/volt. The passive flow of ions through channels is influenced by both a chemical and an electrical gradient. The two forces are balanced at the Nernst potential. The transmembrane potential of a cell is a weighted average of the Nernst potential of each open ion channel. Current passing through an ion channel is measured with a patch-clamp technique. In this technique, an electrode pressed against the cell membrane forms a sealed 1 to 3 um2 area of membrane within which all flowing ions are captured. The subject of ion channels is reviewed by Ackerman and Clapham (1997).
Membrane Hydration
One result of the interaction of ion channels is the capability of normal airway epithelia to either absorb salt and water (driven by active Na transport) or secrete liquid (driven by active Cl transport). Active transport of Na drives liquid absorption in two steps: (1) Na enters the cell through the ENaCs; and (2) Na is pumped from the cell by the Na/K/ATPase on the basolateral membrane. CFTR at least partially determines the normal secretion/absorption balance by at least three of its functions: conduction of Cl- ions, inhibition of ENaC. and stimulation of alternative Cl- channels such as outwardly rectifing chloride channels (ORCCs). Thus, CFIR mutations that both drastically affect its ability to move Cl- effectively itself and also eliminate its ability to regulate other channels cause the most severe disease. The channeling and regulation functions of CFTR are not necessarily tightly coupled; one can be eliminated while the other is preserved. (Schwiebert, Morales et al. 1998).
It is possible to supplant each of the CFTR functions that determine membrane hydration with CFTR-sparing drugs. Drugs that inhibit complementary ion channels, particularly ENaC, can be use to supplant the regulatory function of CFTR over ENaC. Amiloride and triamterene bind to ENaC and block conduction (Barbry, Champigny, et al. 1996). Epithelial Na+ channel activity is tightly controlled by several distinct hormonal systems, including corticosteroids and vasopressin. For example, synthesis of ENaC is positively regulated by aldosterone, and the aldosterone antagonist spironolactone can be used to reduce the number of ENaC channels (Haris and Rado 1996). CFTR regulation of alternative ion channels, such as ORCCs, can be supplanted with purinergic receptor agonists, for example UTP (Knowles, M. R., Olivier, K. N., et al 1995). This is because CFTR regulates ORCCs by facilitating the release of ATP out of cells (Sugita, Yue et al. 1998). Once released from cells, ATP stimulates ORCCs by means of a purinergic receptor P2Y2 (Fulmer, Schwiebert et al. 1995). Finally, CFTR Cl- channel function in mutant CFTR channels can be recovered by several means (see ## MISSING MARKER: xe2x80x9ctable mutant mechanismsxe2x80x9d ##).
Alternative Cl- Channel Activators
Activators of alternative Cl- channels that could spare CFTR function extend beyond ATP and UTP: Ca(2)-ATPase inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone(Chao, Kouyama et al. 1995); 1-ethyl-2-benzimidazolone (1-EBIO) (Devor, Singh et al. 1996); psoralen (Devor, Singh, et al 1997); UDP (Lazarowski, Paradiso, et al 1997); UDP binds to a receptor distinct from P2Y2 (Lazarowski, Paradiso, et al 1997).
Schwiebert, Cid-Soto et al. (1998) have explored the possibility of manipulating ClC-2 chloride channels by pharmacotherapy to relieve the symptoms of CF.
Aerosolized uridine triphosphate (UTP) induces Cl- (and liquid) secretion in CF airway epithelia via non-CFTR Cl- channels. Short-term aerosolized UTP is well tolerated by normal subjects and patients with CF, and pilot studies in normal subjects show that aerosolized UTP is an effective stimulator of mucociliary clearance (Knowles, M. R., Olivier, K. N., et al 1995).
Purinergic Receptors
Purinergic receptors have a significant role in the present invention, and a brief description of these receptors follows. Nomenclature, agonists, and tissue distribution of purinergic receptors are reviewed by Heilbronn, Knoblauch et al (1997). An extended description is also provided in P2 purinoceptors; localization, function and transduction mechanisms, Ciba Foundation Symposium 198. Ed. by D. J. Chadwick and J. A. Goode, pub. John Wiley and Sons (1996). Parr et al (1994) describe the use of the human purinergic (P2U) receptor to bypass the CFTR Cl- secretory pathway. ATP and UTP bind to the receptor, and in airway epithelia, the cellular response is to activate an alternative, non-CFTR-dependent Cl- conductance. Another response to purinergic reception is the secretion of mucus by goblet cells. Other tissues with known P2U receptor function include hepatic biliary cells and thyroid tissue (Heillbronn and Knoblauch, 1997). P2Y2 receptors, UTP-P2Y4 receptors and unidentified ATP-specific receptors are said to be present in human tracheal gland cells (Merten, Saleh et al. 1998). The P2U receptor is described in U.S. Pat. No. 691,156 issued to Boucher et al. Nov. 25, 1997. Purinergic receptor agonists have been disclosed in U.S. Pat. No. 5,641,500 issued Jun. 24, 1997 to Trepel et al., with assertions of improved therapeutic features such as increased resistance to hydrolysis. Examples of these compounds include, but are not limited to, ADP, AMP, AMP-PNP, xcex1,xcex3 methylene ATP, and ATPxcex3S.
Currently, clinical tests on the use of UTP to treat CF symptoms are being conducted. Thus far, ATP has been avoided to avoid the bronchoconstrictor effects of its adenosine moiety. Clinical trials on the use of amiloride and UTP together are also being conducted (Boucher 1994).
Besides the direct effect of purinergic receptors on alternative ion channels, they have other physiological effects that are relevant to the present invention. For example, extracellular ATP promotes cellular proliferation in renal inner medullary collecting duct cells and this occurs through purinergic receptors (Ishikawa, Higashiyama et al. 1997).
Actin filaments directly interact with CFTR and regulate its channel activity (Cantiello 1996; Ismailov, Berdiev et al. 1997; Brezillon, Zahm et al. 1997).
Recently, Wersto et al described the use of a fluorescent dye, dihydrorhodamine 6G (dR6G), to stain cells expressing functional CFTR Cl- permeability. Transfer of the CFTR gene into CFTR- cells increased staining by the dye. The staining pattern tracks expected physiological activity. cAMP stimulates dR6G staining and cAMP antagonists inhibit staining. Staining is ATP- dependent and inhibited by Cl- removal or the addition of 10 mM SCN-. dR6G fluorescence distinguishes amended cells after gene therapy (Wersto, Rosenthal et al. 1996).
Mutants
Selected mutants are discussed below to provide a sense of the spectrum of phenotypes that can result from various mutations in the CFTR gene.
Five mechanisms by which mutations disrupt cystic fibrosis transmembrane conductance regulator function have been suggested by (Kerem and Kerem 1995). They are listed in Table 3, along with possible corrective treatments for each mechanism.
A biochemical arrest in processing the xcex94F508-CFTR mutant protein, the predominant mutation within the human population, prevents CFTR from exiting the endoplasmic reticulum (ER) where it is synthesized (Zhang, Kartner et al. 1998). Although ER-retention mechanisms recognize conformational changes in the mutant protein, they do not necessarily affect other CFTR properties. Both xcex94F508-CFrR as well as CFTR function as cAMP-regulated chloride channels in native endoplasrnic reticulum membrane (Pasyk and Foskett 1995). Moreover, retention within the ER of xcex94F508 is conditional. A CFTR-like Cl- conductance appears after incubation of CF-affected airway epithelial cells at 25-27xc2x0 C. (Egan, Schwiebert et al. 1995). Also, treatment with xe2x80x9cchemical chaperonesxe2x80x9d results in the appearance of a fully glycosylated and mature form of the xcex94F508 CFTR protein at the plasma membrane (Brown, Hong-Brown et al. 1996; Welch and Brown 1996; Sato, S., Ward, C. L., et all996). Other suggestions for rescue of xcex94F508 CFTR protein include phenylbutyrate (Rubenstein, Egan et al. 1997; Rubenstein and Zeitlin 1998). Intragenic suppressor mutations in NBD1 (for example R553 and R555K) allow xcex94F508 to escape from the ER and function as Cl- channels on the cell surface (Teem, Carson et al 1996).
Mutations in CFTR can be associated with elevated sweat chloride concentrations in the absence of the CF phenotype. A 6.8 kb deletion (D14a) and a nonsense mutation (S1455X) in the CFTR genes of a mother and her youngest daughter are associated with isolated elevated sweat chloride concentrations. Detailed clinical evaluation of both individuals found no evidence of pulmonary or pancreatic disease characteristic of CF. mutations in CFTR can be associated with elevated sweat chloride concentrations in the absence of the CF phenotype (Mickle, Macek Jr et al. 1998).
Animal Models
Lansdell, Delaney et al (1998) have conducted a detailed comparison of the mouse and the human CFTR. They conclude that although human and murine CFTR have many properties in common, some important differences in function are observed. These known differences could be considered in analyzing any results obtained from an animal model of cystic fibrosis. Several groups have constructed transgenic mouse models of CF, including a model of the predominant human mutation xcex94508 (Dickinson, Dorin et al. 1995; Clarke, Grubb et al. 1994; Colledge, Abella et al 1995). The CFTR homozygote shows defects in the airway and intestinal epithelia similar to those in the corresponding human tissues. Most of the mutant mice demonstrate meconium ileus pathology. Three models exhibit a very high level of fatal intestinal obstruction. (Zhou, Dey et al. 1994) injected the human CFTR gene into cftr- mouse fertilized oocytes. The mice survived and showed functional correction of ileal goblet cell and crypt cell hyperplasia and cAMP-stimulated chloride secretion.
Delaney et al, created mice (Delaney, Alton et al. 1996) carrying the missense mutation G551D and they reflect the human genotype/phenotype of the mutation. They produce normal CFTR at low levels, do not die soon after birth, and are fertile. These mice were used to study the development of lung disease. Unlike humans, they do not show any gross lung pathology, probably because of inter-species differences in ion channel distribution.
Unlike humans, male CF mice are usually fertile. This has been attributed to the presence in mice of an alternative ion channel (Leung, Wong et al. 1996).
Large animal CF models have also been developed, including a primate model. The nucleotide sequence of the coding region of rhesus CFTR is 98.3% identical to human CFTR and the amino acid sequence is 98.2% identical and 99.7% similar. Flanking introns are about 91.1% identical to human introns (Wine, Glavac et al. 1998).
Infection
Most of the morbidity and mortality caused by CF is due to chronic infection of the airways. Bacteria commonly isolated from CF sputum include Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa (FitzSimmons 1994) . Other pathogens such as Burkholderia (Pseudomonas) cepacia, Stenotrophomonas (Xanthomonas) maltophilia, B. gladioli, Aspergillus fumigatus, and nontuberculous mycobacteria are also problematic in some patients.
Genetic Therapy
Twenty clinical trials of gene therapy for cystic fibrosis have been initiated using viral and non-viral vectors for gene transfer (Marcel and Grausz 1997). Vectors that have been studied in attempts to develop gene therapy for CF include adenoviruses, adeno-associated viruses (AAV), and liposomes (MacVinish, Goddard et al 1997).
Yeast artificial chromosomes represent a promising technology for transferring large amounts of DNA (Ripoll, Cowper et al 1998).
Knowles, M. R., Paradiso, A. M. et al (1995) have proposed a method to measure the biological efficacy of gene transfer of the normal CFTR cDNA into CF respiratory epithelia.
Recently adenoviral gene transfer vectors devoid of all viral coding sequences was used to obtain regulated gene expression with decreased toxicity using genomic DNA for gene transfer (Schiedner, Morral et al. 1998).
Synthetic non-viral vectors have large DNA capacities, and they are relatively non-toxic and non-immunogenic. Recent publications on the design and application of non-viral vectors include (Boasquevisque, Mora et al. 1998); (Byk, Dubertret et al. 1998; Escriou, Ciolina et al. 1998); (Murphy, Uno et al. 1998). An aerosolized vector suitable for use with CF patients is of particular interest (Eastman 1998).
Viral systems being developed for use as vectors for ex vivo and in vivo gene transfer include retroviruses, adenoviruses, herpes-simplex viruses and adeno-associated viruses. (Robbins, Tahara et al. 1998; Gene Therapy Protocols (Methods in Molecular Medicine) by Paul D. Robbins (Editor) Humana Press, 1997).
DOTAP cationic liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis has been shown to be safe and efficacious (Porteous, Dorin et al. 1997).
(Knowles, Noone et al. 1998) have announced a study to evaluate the safety and efficacy of a lipid-DNA complex for producing CFTR gene transfer and correcting the chloride ion transport defect in the nasal epithelium of patients with cystic fibrosis.
Davies has succeeded in correcting the abnormal binding of P. aeruginosa to CF respiratory epithelial cells by transfection with CFTR/liposome complexes (Davies, Stern et al. 1997).
Johnson et al. have assessed the variables affecting the efficiency and efficacy of adenovirus-mediated gene transfer to cystic fibrosis airway epithelia (Johnson, Pickles et al. 1996)
In vivo transfer of purified CFTR protein via phospholipid liposomes into the apical membrane of nasal epithelia of CFTR knockout mice has been demonstrated (Ramjeesingh, Huan, et al. 1998).
As discussed above, in accordance with the present invention, we have demonstrated, where others have failed, that it is possible to conduct gene transfer in a prenatal infant to correct genetic defects in the infant prior to birth. This objective is achieved through the direct delivery of a foreign gene into the amniotic fluid surrounding a prenatal infant mammal inutero. Depending upon the developmental age of the infant when the delivery of the gene is accomplished is essentially determinative of the tissue or tissues in which the gene will be taken up in the cells and expressed therein. In particular, however, we have demonstrated the invention""s extreme benefit in connection with the delivery of foreign genes to the lungs of prenatal infant mammals.
Moreover, the present invention allows, through careful selection from amongst several factors, delivery of genes to cells in prenatal infant mammals in a manner that maximizes expression of the gene and minimizes the potential for an immune response in the infant mammal against the foreign gene. Through this work, we have identified a novel lung cell that has altered phenotype and morphology form other lung cells. Further, such cells excrete or secrete glycoconjugates and/or lipids (and particularly, neutral lipids) that appear to protect or xe2x80x9cimmunoprotectxe2x80x9d the cells, and the lung in general, from opportunistic infections, such as bacterial infections. xe2x80x9cGlycoconjugatesxe2x80x9d as used herein refer to agents which include glycoproteins and glycolipids, as well as potentially other glcosylated agents. Thus, the excretory or secretory products of the cells appear directly involved in immunoprotection of cells and are referred to herein as xe2x80x9cimmunoprotectant factorsxe2x80x9d. As will be appreciated, the mode operation of the xe2x80x9cimmuno-protectant factorsxe2x80x9d could be through bacteriostatic, bacterialcidal, immune stimulatory, antireplicatory, or through other modes or processes. Generally, the term xe2x80x9cimmunoprotectant factorsxe2x80x9d is used to described any mode in which cells or tissues are protected from opportunistic infections.
Such immunoprotectant factors may be absent in patients suffering from cystic fibrosis disease (CF) and would assist in explaining the susceptibility of CF patients to chronic lung infections. Further, as will be discussed in greater detail below, such immunoprotectant factors are expected to be useful for the treatment of far more than the chronic lung infections in CF. Further, the factors are expected to find application in mitigating a variety of bacterial, viral, and other infections.
The present invention encompasses the following specific subject matter:
In accordance with a first aspect of the present invention, there is provided a cell population derived from a population of pleuripotential stem cells in a portion of a mammal""s developing respiratory epithelium, pleuripotential stem cells having been transfected with a viral vector comprising the hCFTR gene and undergone expression of the gene prior to differentiation.
In a preferred embodiment, the viral vector is an adenoviral vector. In another preferred embodiment, the transfection of the pleuripotential stem cells is accomplished in vivo. In another preferred embodiment, the transfection of the pleuripotential stem cells is accomplished in vitro. In another preferred embodiment, the cell population secretes glycoconjugates and lipids that are immunoprotectant to the cell population and mitigate bacterial infection, such as infection with Pseudomonas aeruginosa. 
In accordance with a second aspect of the present invention, there is provided a cell population derived from a population of pleuripotential stem cells in a mammal""s developing epithelium, the pleuripotential stem cells having been transfected with a viral vector comprising the hCFTR gene and undergone expression of the gene prior to differentiation.
In a preferred embodiment the viral vector is an adenoviral vector. In another preferred embodiment, the transfection of the pleuripotential stem cells is accomplished in vivo. In another preferred embodiment, the transfection of the pleuripotential stem cells is accomplished in vitro. In another preferred embodiment, the cell population secretes glycoconjugates and lipids that are immunoprotectant to the cell population and mitigate bacterial infection, such as infection with Pseudomonas aeruginosa. In another preferred embodiment, the developing epithelium comprises respiratory tissues. In another preferred embodiment, the developing epithelium comprises the gut.
In accordance with a third aspect of the present invention, there is provided a method of generating cell populations secreting immunoprotectant factors in a prenatal infant, the infant being surrounded by amniotic fluid in utero. In the method, a gene construct comprising an hCFTR gene is delivered into the amniotic fluid at a time when the infant possesses a population of pleuripotential stem cells in the infant""s developing epithelium the gene construct being delivered in a manner designed to transfect the pleuripotential stem cells with the hCFTR gene, wherein, upon differentiation, at least a portion of the pleuripotential stem cells form cell populations secreting immunoprotectant factors.
In a preferred embodiment, the gene construct comprises a viral vector. In another preferred embodiment, the viral vector is an adenoviral vector.
In accordance with a fourth aspect of the present invention, there is provided an isolated sample of glycoconjugates and lipids secreted by a cell population derived from a population of pleuripotential stem cells in a mammal""s developing epithelium, the pleuripotential stem cells having been transfected with a viral vector comprising the hCFTR gene and undergone expression of the gene prior to differentiation.
In a preferred embodiment, the viral vector is an adenoviral vector. In another preferred embodiment, the transfection of the pleuripotential stem cells is accomplished in vivo. In another preferred embodiment, the transfection of the pleuripotential stem cells is accomplished in vitro. In another preferred embodiment, the cell population secretes glycoconjugates and lipids that are immunoprotectant to the cell population and mitigate bacterial infection, such as infection with Pseudomonas aeruginosa. In another preferred embodiment, the developing epithelium comprises respiratory tissues. In another preferred embodiment, the developing epithelium comprises the gut.
In accordance with a fifth aspect of the present invention, there is provided a method of generating a secretory cell population, the secretory cell population being capable of secreting factors that immunoprotect the secretory cell population from opportunistic infections. In the method, a population of pleuripotential stem cells are transfected with a gene construct comprising the hCFTR gene in a manner designed to cause expression of the hCFTR gene, wherein, upon expression of the hCFTR gene and upon development and differentiation of the pleuripotential stem cell population, the secretory cell population is formed.
In a preferred embodiment, the gene construct comprises a viral vector. In another preferred embodiment, the viral vector is an adenoviral vector. In another preferred embodiment, the transfection of the pleuripotential stem cells is accomplished in vivo. In another preferred embodiment, the transfection of the pleuripotential stem cells is accomplished in vitro. In another preferred embodiment, the factors comprise glycoconjugates and lipids. In another preferred embodiment, the factors that are immuno-protectant to the cell population and mitigate bacterial infection, such as infection with Pseudomonas aeruginosa. 
In accordance with a sixth aspect of the present invention, there is provided a secretory cell population that is derived from a pleuripotential stem cell population after transfection with, and expression of, a gene construct comprising the hCFTR gene and development and differentiation of the pleuripotential stem cell population.
In a preferred embodiment, the gene construct comprises a viral vector. In another preferred embodiment, the viral vector is an adenoviral vector. In another preferred embodiment, the transfection of the pleuripotential stem cells is accomplished in vivo. In another preferred embodiment, the transfection of the pleuripotential stem cells is accomplished in vitro. In another preferred embodiment, the secretory cell population secretes factors comprising glycoconjugates and lipids. In another preferred embodiment, the factors that are immunoprotectant to the secretory cell population. In another preferred embodiment, the factors mitigate bacterial infection, such as infection with Pseudomonas aeruginosa. 
In accordance with a seventh aspect of the present invention, there is provided a pleuripotential stem cell differentiation factor comprising a gene corresponding substantially to hCFTR gene.
In accordance with an eighth aspect of the present invention, there is provided a method to treat opportunistic infections in a mammal, comprising administering an isolated immunoprotectant factor selected from the group consisting of glycoconjugates and lipids in a suitable carrier to affected cells and/or tissues.
In a preferred embodiment, the opportunistic infection is associated with a disease. In another embodiment, the disease is selected from the group consisting of Pneumocystis carinii pneumonia (PCP), cystic fibrosis (CF), acute respiratory distress syndrome (ARDS), necrotizing enteral colitis, SCIDS, ADA, immunosuppressed and diarrheal disease. The immunoprotectant factors can be derived from secretory cell populations as described above.
In a ninth aspect of the present invention, there is provided a composition for protecting cells from opportunistic infections comprising an agent selected from the group consisting of glycoconjugates and lipids, wherein the agent is substantially similar or identical to a second agent isolated from a secretory cell population that is derived from a pleuripotential stem cell population after transfection with, and expression of, a gene construct comprising the hCFTR gene and development and differentiation of the pleuripotential stem cell population.
Our laboratory transiently expressed cftr in utero and permanently corrected the lethal phenotype of the cftrxe2x88x92/xe2x88x92mouse. Some of the results were disclosed by Larson, Morrow et al. (1997). An in utero gene therapy did not permanently replace the cAMP-dependent chloride channel and continuous functioning CFTR was not required for the correction of the lethal intestinal obstruction of the cftrxe2x88x92/xe2x88x92mice. The first animals rescued by in utero CFTR lived for greater than one year old. Their untreated littermates do not survive into adulthood ( greater than 45 days of age). These results suggested that the rescue of the knockout mouse was due to a temporary requirement of CFTR for normal epithelial development in the intestines.
We have devised a way to apply methods that temporarily ameliorate CFTR deficiency to in utero therapy, with the surprising result that amelioration then extends beyond birth and beyond the duration of the temporary in utero effect to become long-lasting amelioration.
Possible means for the brief amelioration includes, but is not limited to, transgenic supplementation of the cftr gene, pharmaceutical stimulation of CFTR, in vivo transfer of purified CFTR protein into the apical membrane of nasal epithelia, and rescue or stimulation of latent CFTR protein molecules. For the purposes of the present invention, latent CFTR protein molecules are understood to be CFTR protein molecules that are from a mutant CFTR gene, functionally defective in any way, retained within the endoplasmic reticulum, or present in insufficient functional quantity. The present invention also includes, but is not limited to, methods for sparing CFHR function such as: pharmaceutical manipulation of alternative ion channels; transgenic manipulation of the expression of alternative ion channels; pharmaceutical manipulation of complimentary ion channels, for example the ENaC channel; and transgenic manipulation of complimentary ion channels, such as the ENaC channel.
One mode of the present invention comprises in utero treatment of an animal to temporarily mitigate the condition of CFTR deficiency by transgenic therapy, with the surprising result that the animal subsequently is relieved of many or all of the symptoms of cystic fibrosis. The treatment may comprise transfer of the CFTR gene by viral vector, liposome or other medium. Many examples of methods of genetic transfer are mentioned herein, and any of these as well as other methods of gene transfer not specifically mentioned, can be used to implement in utero gene therapy with long-lasting improvement on the symptoms of cystic fibrosis.
The present invention also may comprise drug therapy, such as with ATP, UTP, or other purinergic ligands; chemical chaperones, choride conductance stimulators such as xanthines and fluoride; inorganic pyrophosphates as disclosed in U.S. Pat. No. 5,686,114, issued to M. J. Welsh, Nov. 11, 1997, and complimentary channel regulators such as amiloride, triamterene and spironolactone. Other possible therapeutic agents are discussed herein and tabulated in Table 2 and Table 3; one of those listed could be utilized in the present invention. In addition, U.S. Pat. No. 5,620,676 issued to Jacobson; et al Apr. 15, 1997 discloses novel adenosine triphosphate (ATP), xanthine and uracil analogs, and these also could be used in the present invention.
Any compounds not explicitly mentioned herein could be utilized in the invention if its in utero use is sufficient to at least temporarily ameliorate the CFTR deficiency present in a patient. The inventors provide examples of dosages and times of treatments; and other ways to vary these conditions are known to skilled workers. Pharmaceutically acceptable carriers are materials useful for the purpose of administering the medicament, which are preferably sterile and non-toxic, and may be solid, liquid, or gaseous materials, which are otherwise inert and medically acceptable, and are compatible with other active ingredients. The pharmaceutical compositions may contain other active ingredients as preservatives. Pharmaceutical dosage forms and drug delivery systems are described by H. C. Ansel, N. G. Popovich and L. V. Allen, Jr. in Pharmaceutical dosage Forms and Drug Delivery Systems, 6th ed. Williams and Willkins (1995).
Relief from or alleviation of symptoms may comprise resistance to infection by any organism, but especially by those organisms known to infect CF patients, for example Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, Burkholderia (Pseudomonas) cepacia, Stenotrophomonas (Xanthomonas) maltophilia, B. gladioli, Aspergillus fumigatus and nontuberculous mycobacteria, and most especially by Pseudomonas aeruginosa; changes in histological staining of tissue, including but not limited to, the stains mentioned herein; gastrointestinal problems such as obstructions and the like; improved respiratory function; and improved function of exocrine glands of the intestine or bronchus. Other symptoms not explicitly mentioned may also be relieved.
A surprising aspect of this invention is the possiblity that over-dosing of the cftr-treatment and/or other in utero therapy targeted to temporarily mitigate cftr deficiency may result in an overcorrection. The over-correction is manifested as hyperplastic growth within the lung. Thus, the effective dose may be determined by any means appropriate for detecting hyperplastic tissue. Description of the use of such histological techniques is provided in Advanced Histopathology by Gordon W. H. Stamp, N. A. Wright, Springer Verlag, 1990; and a Textbook of Histologyxcx9cby Bloom and Fawcett, 12th Edition Chapman and Hall, 1994. In particular, we have shown that the application of HandE stain to lung sections reveals the extent of hyperplasia. Thus, use of the HandE stain on respiratory tissue, as well as other means for detecting hyperplasia, would allow a person skilled in the art to optimize the therapeutic dose and timing of its administration.
As used herein, the term xe2x80x9ceffective dosexe2x80x9d is denoted to mean a predetermined amount sufficient to ameliorate a CFTR deficiency in utero, with the amelioration extending past the subsequent birth of the treated subject. Manifestations of CFTR deficiency are usually described as cystic fibrosis, but other terms are used, for example congenital absence of vas deferens (CAVD).
Another mode of the present invention is to apply our observations for diagnostic/prognostic purposes, and for monitoring the progress of therapy. These observations include the use of stains to monitor the conditions of tissue. In addition to the histological texts previously mentioned, Molecular Probes, Inc. of Eugene, Oreg., USA, has published a comprehensive manual by R. P. Haugland that describes the use of fluorescent stains: Handbook of Fluorescent Probes and Research Chemicals, 6th ed (1996). In particular, histological stains include the use of means to detect changes in intracellular Ca++ after application of the therapy of the present invention. Means of detecting changes in intracellular Ca++ would include particularly the use of Ca++ specific stains and the use of Ca++ specific sensors, including but not limited to, the one described by Shalom et al (Shalom, Strinkovski et al. 1997). The Shalom sensor is based on a pulled micropipet, filled with a conducting porous sol-gel glass which has been doped with the fluorescent Calcium Green(trademark) Ca2+ indicator. Such sensors are potentially capable of measuring Ca2+ concentrations as low as 10(xe2x88x928) M, in confined volumes, with a three-dimensional resolution which exceeds approximately 0.1 micron. Other techniques for measuring Ca++ and Ca++ flux are described in Methods in Cell Biology, vol. 40; ed. R. Nuccitelli, Academic Press (1994). Methods described in this text include microelectrode techniques, including calcium-specific electrodes (Chapter 4, Baudet et al) vibrating electrodes (Chapter 5, Smith et al), and patch clamp methods (Chapter 6, Leech and Holz); fluorescene techniques for imaging Ca++ (Part III); and the use of aequorin for Ca++ imaging (Part IV). Fluorescence techniques for Ca++ imaging include the use of a variety of indicator for Ca++, for example Calcium Green-1(trademark), Calcium Green-2(trademark), Calcium Green-5N(trademark) fluo-3, indo-1, rhod-2, fura-2, fura red, indo-1, fura(-2)dextran and indo(-1)dextran. Appropriate histological techniques also include the use of ligands to stain purinergic receptors. Thus, use of these and similar physiological stains would allow a person skilled in the art to optimize the dose and timing of a therapy targeted to mitigating a CFTR deficiency in utero, particularly when used in conjunction with a stain for hyperplasia.